The cytoplasmic membrane of E. coli contains an active protease. This protease degrades the intrinsic membrane protein nitrate reductase, and, under certain conditions, other membrane proteins as well, both in situ and in isolated washed membrane. The purpose of this project is to investigate the protease(s) which cause this degradation. Since degradation of both normal and mutationally-altered nitrate reductase occurs under several different sets of conditions, the fragmentation pattern and protease inhibitor sensitivity under these different conditions will be compared to see if more than one protease is involved. In these and other experiments, the degraded enzyme is isolated by immune precipitation. Both normal and inverted membrane vesicles will be surface labeled and allowed to undergo proteolysis, to determine if the protease is side-specific and if it will preferentially release proteins accessible to the surface. An attempt will be made to label the protease(s) with labeled protease inhibitors. Degradation of nitrate reductase in situ will be examined under several different conditions to determine the physiological role of the protease. The proteolytic activity of the membrane can be solubilized with Triton X-100, and an attempt will be made to purify the protease by detergent column chromatography and characterize the purified enzyme (or enzymes) which is obtained.